Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein
- Naoko Sasabe†1,
- Yuka Keyamura†1, 2,
- Takashi Obama1,
- Nozomi Inoue1,
- Yukihiro Masuko1,
- Yu Igarashi1,
- Toshihiro Aiuchi1,
- Rina Kato1,
- Tomohiro Yamaguchi1,
- Hiroshi Kuwata3,
- Sanju Iwamoto4,
- Akira Miyazaki4,
- Shuntaro Hara3,
- Tomohiro Yoshikawa2 and
- Hiroyuki Itabe1Email author
© Sasabe et al.; licensee BioMed Central Ltd. 2014
Received: 8 February 2014
Accepted: 6 March 2014
Published: 14 March 2014
Oxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced.
Human and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry.
When human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL.
These results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.
KeywordsOxidized LDL PC molecular species LC-MS/MS Oxidized PC PAF-AH Pefabloc lysoPC PONPC Rabbit LDL
Oxidative modification of low-density lipoprotein (LDL) is involved in atherosclerotic lesion development [1–3]. Studies have shown significant increases in plasma circulating oxidized LDL (oxLDL) in patients with cardiovascular diseases [4–8]. LDL consists of phospholipids, cholesterol ester, triacylglycerol, and an apolipoprotein B (apoB) together with several minor constituents, so that oxLDL may contain a large variety of oxidized products and modified molecules [9–11].
Many biological and pathological activities of oxLDL have been investigated, and oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) formed during oxidation of LDL are thought to be responsible for some oxLDL functions. OxPC is a mixture of diverse products characterized by various functional groups, including both long chain acyl groups with hydroperoxide or hydroxyl function and truncated short chain acyl groups. Macrophage scavenger receptors take up oxLDL leading to foam cell formation, where oxPC is involved in recognition by scavenger receptors [12, 13]. OxPC could act as a peroxisome proliferator activating receptor (PPAR)-γ activator to induce inflammatory and metabolic responses . It promotes chemokine production in endothelial cells [15, 16] and proliferation and calcification of smooth muscle cells . In addition, oxPC induces inflammatory reactions by acting as an environmental pathogen mimetic . OxPC, especially PC hydroperoxides (PCOOH), increases macrophage adhesion to ICAM-1 through reorganization of microfilaments . LysoPC is another atherogenic metabolite formed in oxLDL that shows many biological effects including attenuation of endothelial functions and proliferation of smooth muscle cells [20, 21].
OxPC is further modified chemically or enzymatically in plasma. Several enzymes metabolize oxPC, including phospholipid hydroperoxide glutathione peroxidase (PHGPx) , secretory phospholipase A2 type IIA (sPLA2-IIA) , lecithin-cholesterol acyltransferase (LCAT) , and platelet-activating factor acetyl hydrolase (PAF-AH/Lp-PLA2) . These enzyme activities make oxidized PC profiles more complex.
PAF-AH is a unique plasma phospholipase that hydrolyzes PC species containing short and/or hydrophilic acyl chains as substrates in addition to PAF itself [25, 26]. It is considered that PAF-AH contributes substantially to the detoxification of pro-atherogenic oxPC products into lysoPC. Steinbrecher reported that the phospholipase A2 activity for short-chain PC substrates in human LDL was not separated from PAF-AH . The enzyme associates with lipoprotein particles, LDL and high-density lipoprotein (HDL), in human plasma  and is found primarily in association with HDL in rabbit and rodent plasma [28, 29]. Because PAF-AH has an active serine residue in its catalytic center, enzyme activity is inhibited by serine-protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF). Pefabloc, a sufonylfluoride compound similar to PMSF, does not inhibit paraoxonase-1, LCAT, or venom phospholipase A2, has been used as a selective inhibitor of PAF-AH [30–32].
Although the pathological impact of oxLDL is recognized, neither the complete structure of oxLDL nor the molecular process of oxLDL generation has been fully determined. Recently liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been applied to the comprehensive analysis PC molecular species including oxidized products [33–35]. Rabbits have been used for diet-induced atherosclerosis experiments, although many animals including rodents are not susceptible to atherosclerosis without genetic manipulation. Checking PC profiles of human and rabbit oxLDLs could give an idea for atherogenic oxPC products. In the present study, we investigated the PC profile changes during copper-induced oxidation of LDL to elucidate the process of PC modification by LC-MS/MS. We also investigated the contribution of PAF-AH to PC profile changes using a PAF-AH inhibitor and rabbit LDL in addition to human LDL.
PC profile change in human LDL during oxidation
List of 42 PC species monitored by LC-MS/MS
Long chain oxPC
34:2 + 14
34:2 + 16
34:2 + 32
36:4 + 14
36:4 + 16
36:4 + 32
36:2 + 14
36:2 + 16
36:2 + 32
38:4 + 14
38:4 + 16
38:4 + 32
PC profiles in rabbit LDL during oxidation
Next, we examined changes in the PC profile in rabbit LDL during oxidation. LDL was prepared from New Zealand white rabbits fed normal diet supplemented with 0.5% cholesterol to increase LDL content in plasma. The PC profile of rabbit LDL is substantially different from that of human LDL. Rabbit LDL is relatively rich S/MUFA-PC and lysoPC. Rabbit LDL was needed a longer incubation than human LDL to reach the maximum oxidation, which may be caused by lower content of PC species with more than five double bonds. PAF-AH activity in the rabbit LDL fraction was estimated to be 2.9 nmol/min/mg, which is 25% of that in human LDL. The PAF-AH activity in the LDL fraction was 6.0% of HDL fraction recovered from the same rabbits, which corresponds to a previous report that PAF-AH is present mostly in HDL in rabbit plasma and very little in LDL . Pretreatment of the rabbit LDL fraction with pefabloc reduced PAF-AH activity by 91%.
Protein modification with oxPC
OxLDL is thought to be an important factor in atherosclerosis. Evidence is accumulating that the plasma level of in vivo oxLDL is a predictor of cardiovascular disease [4–8]. Previous studies revealed that many atherogenic oxPCs and lysoPC are formed in oxLDL [12, 15, 18, 20]. However, the manner in which LDL is oxidatively modified remains to be determined. In this study we analyzed the time-course changes in the PC profile during LDL oxidation using LC-MS/MS to view the full picture of oxPC generation and the contribution of PAF-AH. Our study shows that PAF-AH has a crucial role in generating lysoPC during LDL oxidation. Many cleaved oxPC and long-chain oxPC species are generated, but the data show only a few major oxPC species accumulating in oxLDL.
By simultaneously and selectively detecting 42 PC species in a sample using MRM mode of LC-MS/MS, we followed changes in PC profiles during copper-induced oxidation. Because standards for all of the PC species were not available, absolute quantitation of the PC profiles cannot be completed. The data are shown as the relative peak areas of the PC species after normalizing on the basis of internal standard. As expected, most of the PUFA-PC species declined sharply and some of them reduced largely, whereas S/MUFA-PC did not change. Long-chain oxPC with four double bonds were unstable and appeared transient. Interestingly, only a few oxPC species accumulated in fully oxidized LDL in vitro, namely PONPC (m/z = 650.6) and mono-oxygenated forms of linoleate-containing PC. It is likely that oxPC species derived from PC containing more than four double bonds are so susceptible to further modification either by chemically or enzymatically that they cannot accumulate in oxLDL.
PCOOH, detected as long chain oxPC species with m/z +32, was present in LDL but was not the major products in oxLDL (Figures 2, 3, 4, 5, 6 and 7, and Additional file 1 for enlarged graphs). Kinoshita, et al. determined the PCOOH concentration in human plasma from healthy control was 160 nmol/L using HPLC with chemiluminescence detector . PCOOH concentration increased to approximately 2-fold in plasma from patients with hyperlipidemia, however, the ratio of PCOOH in total PC was still 1/7,000. Our data cannot directly transfer to quantitative calculations, but relatively small peaks for PCOOH in the human and rabbit LDLs among all the PC species (Figures 2 and 5) corresponds well to the previous study.
LysoPC appeared to be the major product in oxLDL. Its generation was strongly suppressed by pefabloc treatment of both human and rabbit LDL, suggesting that PAF-AH activity is critical for lysoPC generation in LDL. It is well known that PAF-AH can act on PC species with hydrophilic short chain acyl groups, in addition to PAF, to produce lysoPC . Thus it is speculated that pefabloc treatment of LDL increases some oxPC species that are substrates of PAF-AH during oxLDL formation. The peak areas for several cleaved oxPC species further increased by the pefabloc treatment (Figures 4D and 7D). However, the major oxPC species that accumulated in pefabloc-treated LDL during oxidation was almost the same as that of oxLDL with active PAF-AH. Our observation agrees with a previous report by Davis et al., in which an oxPC profile in LDL oxidized with copper sulfate for 20 h was analyzed .
In addition to hydrolysis of cleaved oxPC species, the protective role of PAF-AH in oxLDL modification was suggested by oxPC-apoB adduct formation. Some chemically active oxPC may react with proteins to form adducts, and extensive hydrolysis of oxPC by PAF-AH is protective against apoB modification by oxPC products.
LysoPC generation was largely inhibited in pefabloc-treated oxLDL, but still a very small increase in lysoPC remained. A possible explanation for this observation is non-enzymatic hydrolysis of oxPC. Choi, et al. reported that lysoPC can be generated through spontaneous deacylation of cleaved-chain oxPC products such as 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-PC . Alternatively, other oxPC-hydrolyzing enzymes may distribute in part to LDL and contribute to lysoPC formation. We propose that most of the oxPC species generated are not only hydrolyzed by PAF-AH but also further modified or decomposed in PAF-AH-independent manners.
Inhibition of PAF-AH in apoE-knockout mice resulted in reduction in atherosclerotic lesion size . However, another study reported that adenoviral overexpression of PAF-AH prevents injury-induced neointima in apoE-knockout mice . A PAF-AH inhibitor has been investigated in clinical trials and the prevention of expansion of necrotic core lesions in human was shown, however, the roles of PAF-AH in atherogenesis and oxLDL modification remain uncertain . Our study suggests that PAF-AH inhibition decreased lysoPC formation but had little effect on oxPC accumulation. To understand the effects of PAF-AH on atherosclerosis, the pathological roles of atherogenic oxPC and lysoPC should be elucidated.
It should be noted that the current study focused on oxLDL prepared in vitro. This study is an important step toward understanding the complex nature of oxidized lipoproteins. Elucidation of the contribution of oxLDL to atherogenesis awaits further lipidomic and proteomic studies to characterize the features of circulating oxLDL.
We determined the PC profile of oxLDL using a lipidomic approach. The major products in copper-induced oxLDL were lysoPC species, and PAF-AH plays a critical role in lysoPC generation in LDL. Although many oxPC species are generated, PONPC and long-chain oxPC species derived from linoleate-containing PC are the major products accumulating in oxLDL. These observations shed light on the manner in which oxPCs are modified and metabolized.
Preparation and oxidation of human LDL
Human LDL and oxLDL were prepared as described previously . This study was approved by the Ethical Committee of Showa University. In brief, plasma was separated from human blood from healthy volunteers by centrifugation at 2,000 rpm for 10 min, after which EDTA was added to the plasma (final concentration 0.25 mM). LDL was separated from the human plasma using sequential ultracentrifugation, with addition of KBr to adjust the density, and then dialyzed against PBS containing 0.25 mM EDTA to remove the KBr. The protein concentration of the LDL fraction was determined by the BCA method using BSA as standard.
To inhibit PAF-AH activity of LDL, an aliquot of the LDL fraction (1 mg/mL) was incubated with pefabloc (Boehringer, 0.5 mM) at 37°C for 30 min . The LDL was then passed through a PD-10 desalting column (BioRad) to remove excess pefabloc. The PAF-AH activity was estimated using a PAF-AH assay kit (Cayman Chemicals).
Either LDL or pefabloc-treated LDL (0.2 mg/mL) was incubated with CuSO4 (5 μM) at 37°C for up to 3 h. At the end of the incubation period, 1 μL of 250 mM EDTA was added to stop copper-induced oxidation. Oxidation reaction was evaluated by measurement of TBARS and agarose-gel electromobility assay. After mixtures of 100 μL of sample and 200 μL of TBA reagent (0.375% TBA, 15% trichloroacetic acid, and 0.25 M HCl) were boiled for 15 min, absorbance was measured at 535 nm . Electromobility of oxLDL was evaluated on a 0.5% agarose gel using barbiturate buffer at pH 8.6. Determination of immunoreactivity to an anti-oxPC monoclonal antibody and an anti-apoB polyclonal antibody was performed by sandwich ELISA as described previously .
Preparation and oxidation of rabbit LDL
New Zealand White rabbits (6–7 weeks old) were purchased from Kitayama Labes Co., Ltd. (Nagano, Japan). The study protocol was approved by the Animal Care Committee (permit no. 11–0221) and carried out in accordance with the “Guideline for Animal Care and Use at the Otsuka Pharmaceutical Co. Ltd.”. The rabbits were acclimated and fed a cholesterol-enriched diet containing 0.5% cholesterol (w/w) in a standard rabbit chow (RC4; Oriental Yeast Co. Ltd.) at 80 g/day for 10 weeks as previously described . Blood was collected from the inferior vena cava in EDTA-treated syringes under pentobarbital anesthesia. Then LDL fraction was collected from plasma by centrifugation, 0.25 mM EDTA was added, and LDL was stored in tubes filled with argon gas in a refrigerator. After LDL was dialyzed against PBS in a refrigerator, 5 μM CuSO4 was added to 100 μg/mL of LDL and incubated for up to 8 h at 37°C. The oxidation reaction was stopped by addition of EDTA (final 0.25 μM) to the solution. The oxLDL was stored in tubes filled with argon gas at -80°C.
LC-MS/MS analysis of oxPC
The profile of PC molecular species in LDL and oxLDL was determined by LC-MS/MS (AB SCIEX, QTRAP5500 equipped with Shimadzu LC-10A). LC separations were performed on an Inertsil SIL-100A column (2.1 × 150mm, GL Science, Tokyo, Japan) with elution solvent of acetonitrile/methanol/3% formic acid (18/11/8 (v/v/v)) at a flow rate 200 μL/min as described by Morishita et al.. The synthetic PC standards, didecanoyl-PC, 1-pentadecanoyl-2-lysoPC, PONPC were purchased from Avanti Polar Lipid Inc. Total lipids were extracted from 10 μg of oxLDL by the Bligh and Dyer method . To the recovered chloroform phase, didecanoyl-PC (20 pmol) was added as internal standard. The samples were dried under N2 gas and the dissolved in 200 μL of the elution solvent. For each analysis, 10 μL of the sample was injected. PC species were detected and quantified using MRM procedure, in which 42 selected ions releasing a fragment ion of m/z = 184 corresponding to a phosphorylcholine group were detected.
Data are presented as means ± standard deviation (SD). For the statistical analysis, one-way-ANOVA was carried out, and a p value < 0.05 was taken to be significant.
- cleaved oxPC:
oxPC with truncated acyl chains
Enzyme-linked immunosorbent assay
Liquid chromatography-tandem mass spectrometry
- long-chain oxPC:
oxPC with oxidized acyl chains
Monounsaturated fatty acids
Oxidized low-density lipoprotein
Platelet-activating factor acetylhydrolase
Polyunsaturated fatty acids
PC with polyunsaturated fatty acids
PC with saturated or monounsaturated fatty acids
Thiobarbituric acid-reactive substances.
We thank Drs. Junken Aoki and Aska Inoue in Tohoku University for valuable suggestions and help for lipidomic analysis. This work was supported in part by grants-in-aid from the Ministry of Education, Technology, Sports, Sciences and Technology of Japan (MEXT), grants for the Private University High Technology Research Center Project from MEXT, and Research on Publicly Essential Drugs and Medical Devices from Japan Health Sciences Foundation. We thank Crimson Interactive Pvt. Inc. for editing service of English expressions.
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